Microtubules are an essential part of the structural and organizing machinery in eukaryotic cells, and a complete understanding of their function could potentially lead to new or more effective approaches in the treatment of a variety of common diseases such as cancer, Alzheimer's or Parkinson's. Toward this end, microtubules will be studied using atomic force microscopy (AFM) with the objective of describing microtubules as heterogeneous structures. AFM will be used for a combination of high-resolution structural characterization and local mechanical characterization, and the relationships between molecular-scale heterogeneity and biological function will be examined. The focus will be on two distinct, biologically-relevant microtubule systems: microtubules that are heterogeneous with respect to nucleotide ligand state, and microtubules with the associated protein tau. Heterogeneous microtubules containing discrete regions of GDP and GTP will be studied using a slowly-hydrolyzable analog of GTP, GMPCPP, to create segmented microtubules. A detailed study of the differences between the GDP and GMPCPP regions will be undertaken. Particular attention will be paid to the interface between GMPCPP and GDP regions to examine how structural changes and lattice strain are accommodated across the interface. The second microtubule system that will be studied is microtubules with the associated protein tau, an important microtubule associated protein that promotes growth and prevents depolymerization of microtubules. Tau binding will be examined as a function of assembly conditions to assess the relative importance of two putative binding sites. The different binding sites will be examined to determine how tau affects microtubule mechanics and these data will be related to the biological function of tau in each binding site. This project will provide many new opportunities to evaluate the functions of microtubules because it combines molecular-level structural and mechanical characterization, examines microtubules as heterogeneous and complex structures, and studies microtubule systems that are physiologically relevant. [unreadable] [unreadable] [unreadable]